The overall aim of this new component for INIA is to understand the neurobiology of excessive alcohol consumption. Epidemiological data demonstrate that early initiation of drinking, and particularly excessive drinking, is a major risk factor for eventual alcoholism. Our hypothesis is that both innate differences in gene expression and differences in response to alcohol, particularity to heavy exposure to alcohol in a binge drinking paradigm, contribute to the development of excessive alcohol drinking. We hypothesize that alcohol exposure will affect DNA methylation which will have lasting effects on gene expression and splicing in a way that increases long-term vulnerability to excessive alcohol drinking. All three Specific Aims will be closely linked, by examining gene expression and splicing, DNA methylation, and microRNAs in a coordinated way on the same tissues, so that we can elucidate mechanisms by which alcohol exposure exerts its effects. Our strategy is to study both alcohol-naive animals from lines selected for difference in alcohol preference, to find pre-existing genetic differences that predispose to excessive drinking, and also to study animals that have experienced repeated binge drinking from postnatal day 30 to 58 to determine how that affects gene expression and DNA methylation, and whether the differences persist. We will analyze differences between exposed and unexposed animals at the end of the repeated binges and also 30 days later to see which differences persist. We will focus on key INIA regions: the shell of the nucleus accumbens (Nac-shell), the central nucleus of the amygdala (CeA), and the ventral tegmental area (VTA); we have microarray data from adult males that show many significant differences in gene expression in these regions. Differences found in global studies, and selected candidate genes from human studies, will be tested to determine whether they are controlled in cis. We will study gene expression, splicing, DNA methylation and microRNAs in the same tissues, to allow an integrated, systems-wide understanding of the effects of alcohol that might underlie excessive alcohol consumption. These mechanistic studies cannot be carried out in humans because we can neither control alcohol exposure nor access brain tissue. For that reason, an additional translational component will be to compare the effects of ethanol on key brain regions with those in an accessible tissue, blood, looking forward to potential human studies.